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Biomolecular Electronics
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Biomolecular Electronics
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CHAPTER OUTLINE 1.0 What is biomolecular electronics? ...........................................................1 1.1 Proteins and biomolecular electronics......................................................3 1.2 Proteins and planar devices.....................................................................6 1.3 The future of biomolecular electronics ...................................................10 1.4 A novel idea: electrical control of biomolecular systems .........................12 1.5 References ............................................................................................15
1.0 What is biomolecular electronics? With the expression “biomolecular electronics” we refer nowadays to that branch of technology that exploits molecules or molecular systems of biological origin for interaction with modern electronics. The idea of using biomolecules to assemble hybrid electronic devices stems from molecular electronics (Aviram & Ratner, 1974) and, as such, is intimately connected with the advent of nanosciences and nanotechnologies, dating back to the beginning of the last decade of the past century. The interest in biomolecules stems not only from their size, which is typically in the nanometer range (at least in two spatial dimensions). Rather, it is often connected to a particular standpoint from which one can regard them. Indeed, an interesting point of view is that of regarding biomolecules in general and proteins in particular as self-contained, nanometer-sized functional units that are highly specialized and efficient in performing a certain functional task. Their efficiency in performing a particular activity is traced back to the fact that biomolecules, being parts of living beings, have naturally evolved over billions of years, reaching the current “optimal” degree of specialization for accomplishing a given task. One can often come across this opinion reading the specialized literature or attending topical conferences; however, it appears quite questionable in light of a slightly less naı¨ve understanding of the Theory of Evolution (Fodor & Piattelli-Palmarini, 2010); nevertheless, it is perhaps a good enough starting point to understand the historical motivations which led to the Biomolecular Electronics. http://dx.doi.org/10.1016/B978-1-4557-3142-8.00001-7 Copyright © 2014 Elsevier Inc. All rights reserved.
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remarkable interest shown by a sizable part of the interdisciplinary scientific community in the use of biomolecules for assembling electronic devices. Another element motivating the interest in proteins as components of electronic circuits is their sizes. Indeed, these molecules often configure self-consistent, functional units with typical sizes in the nanometer range. Therefore, it is conceivable to imagine functional devices as small as a single molecule, especially in view of the fantastic progresses in miniaturization and parallelization made with nano-lithographic techniques. Furthermore, the rich chemistry that particularly characterizes proteinforming monomers, i.e., the 20 natural amino acids, makes these molecules amenable to various kinds of chemical functionalization, thus improving their reactivity towards desired functional groups and enabling, as a consequence, their ready chemisorption on pre-functionalized structures and surfaces. Moreover, the exceptionally fine-tuned recognition properties of biological structures (e.g., complementary nucleic acid strands, DNA-binding proteins, antibody-antigen or receptor-ligand pairs, etc.) could allow for selfassembling of more complex structures in the nanometer-sized gap between meso/nanoscale electrodes. Among the various classes of molecules of biological origin, two of them have been especially focused upon by researchers in the field: DNA and proteins. The reasons for this choice are different; in the case of DNA, its robustness and ease of handling are relevant factors. Nevertheless, a further important aspect fostering DNA as an interesting molecule in biomolecular electronics stemmed from the idea that, if we could understand the mechanism by which electrons are driven along the double helix during DNA repair of damage caused by oxidative stress, we would obtain, as a byproduct, a way to produce molecular wires, that is, one-moleculethick conducting wires, to be used in assembling (bio)molecular circuitry. If DNA can be regarded in terms of molecular wire, what else does one need to assemble molecular circuits? Some functionally relevant electronic elements, of course. Those were identified as the proteins, which are indeed deputed to perform the main functional tasks in the organisms that express them. At this point a relevant question arose that has not been completely answered yet, about the electrical conductivity of biomolecules. It is indeed obvious that at the basis of any electronic circuit there is a need to deal with wires and elements that can conduct or, more generally, interact functionally with electron flows, i.e., electron currents. Whereas this issue is still open in the case of DNA, which displays good conductivity over short ranges (of the order of nanometers) and appears to be
1.1 Proteins and biomolecular electronics
an insulator over larger distances (Guo et al., 2008), the situation regarding proteins is more complex and requires detailed analysis because of the large variability of their properties (mirroring the differences in protein structures). In the case of DNA, its poor conductivity, at least in the mesoscopic range, has prompted various approaches for enhancing it, based on doping (with metal ions or intercalating agents (Ban et al., 2009)) or on the use of DNA as a molecular template for metal nanowire growth (Berti et al., 2005). In the case of proteins, which possess an intrinsically higher chemical and structural variability compared with DNA, it is not surprising that specific transport proteins, carrying either electrons or ions, soon attracted the interest of researchers.
1.1 Proteins and biomolecular electronics Typically, electron transport proteins are water-soluble molecules (rare exceptions apart; see, e.g., cytochrome b561, a transmembrane three-heme cytochrome), whereas ion transport proteins are membrane molecules requiring specific environmental conditions (incorporation in a lipid bilayer) to be functionally active (see section 3.6.1). According to this difference, whereas the former are more easily implemented in electronic circuits, the latter are more demanding to exploit in a nano-circuit configuration. Ion channels have been used to gate transistors in hybrid devices thanks to their selectivity for specific ions (Bernards et al., 2006) and the inherent similarity of the gating mechanism of voltage-gated ion channels to the basic features of a field-effect transistor (Bezanilla, 2005). A specific application of ion channels in electronic circuits for sensing applications is the coupling of the channels reconstituted in lipid bilayers with other electronic nanostructures such as semiconductor nanowires (Misra et al., 2009). Examples of these applications are the possibility, by changing the pH of the solution, of controlling the conductance of a silicon nanowire covered with a lipid bilayer integrating peptide pores and the creation of nanobioelectronic transistors exploiting ion pumps such as Naþ/Kþ-ATPase (Huang et al., 2010). However, the most popular and charming class of proteins that seemed to be particularly suitable for implementing electronic devices is the redox metalloproteins. These very important molecules are indeed able to shuttle electrons between molecular partners by reversibly changing the oxidation state of the metal ions they contain (see sections 4.1, 4.1.1, and 4.1.2). Following the route defined by conventional solid-state electronics, the research efforts in biomolecular electronics have generally focused on
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demonstrating transistor-like behavior that proteins might display when positioned between two electrodes. However, considering the classic definition of a transistor, we should stress here a different meaning for the same word. A transistor represents a device composed of at least three electrodes (source, drain, and gate) which is able to amplify or switch the current between a pair of electrodes (e.g., source-drain) by controlling the voltage or current between two of these three electrodes (e.g., source-gate or drain-gate). In conventional electronics the controlling signal is an electric signal. In the context of proteins, among the possible signals that can function for gating a hybrid device we include also other signals of a chemical or physical nature. Among them one has to consider all the parameters that are able to modify, at constant bias voltage, the transport properties of a protein or of a protein layer sandwiched between two electrodes. In a similar way, protein conformational variations can induce changes in its transport properties or in those of another nanostructure to which the protein is coupled. Among these signals one can list variation in environmental pH, temperature, substrate recognition by enzymes and so on. The sensitivity to any of these signals confers sensing properties to the related devices. We will consider the main results obtained along the pathway of understanding the behavior of redox metalloproteins when used as active parts of hybrid electronic devices in Chapter 4. Here, we continue by noting that other features of proteins have also attracted researchers’ attention and have been used in various attempts to exploit the properties of natural biomolecules to implement electronic devices. There is, for instance, the case of antibodies. These molecules are part of an organism’s immune system and are known for their specific recognition and binding capabilities against the corresponding antigens. Their use has been recently demonstrated in assembling single-protein devices (Chen et al., 2012). Particularly, antibodies of the IgG type (immunogammaglobulin) have been raised against Au nanoparticles, 5 nm in diameter. An IgG molecule can bind up to two of these gold nanoparticles, one per Fab fragment, where the specific recognition sites for antigens are located. This specific binding provides a stable bond. Gold nanoparticles are attached to e-beam lithography-fabricated 10-nm gap electrodes that act as source and drain electrodes. Once the electrodes have been decorated with nanoparticles, they are exposed to IgGs that specifically recognize the nanoparticles, binding them firmly. This results in a configuration that can be gated electrically, by means of a proximal electrode, and optically, by the action of a CdSe Qdot attached to the IgG molecule at its Fc fragment. While irradiated, the Qdot absorbs light, providing an induced dipole moment
1.1 Proteins and biomolecular electronics
(confined exciton) that operates an effective gating of the single-protein transistor, probably mediated by conformational modifications occurring in the biomolecule. A richness of features is observed, including current values of the order of a few hundreds of picoamps, rectification, and negative differential resistance (NDR), which is ascribed to the presence of the Fc fragment. Such an exciting implementation is surprising because it shows it is possible for an electric current to flow through a large biomolecule that, physiologically, is not thought to have any electron transport capabilities. This fact raises questions about protein conductivity and, hence, about the suitability of proteins as materials for electronics. Whereas theoretical considerations about protein conductivity and electron transfer in general will be dealt with later in this book (see Chapter 4), systematic experimental studies (Ron et al., 2010) suggest that electron transport through proteins, even measured in the solid state, is more effective than through a generic insulating material. There, the peculiar conductivity displayed by redox metalloproteins is confirmed, but even proteins without a physiological role in electron transfer (e.g., albumin, bacteriorhodopsin) display electron transport ˚ 1 that denote them as promising decay constants b in the range 0.12e0.27 A materials for bioelectronic applications. Indeed, with the same molecule, bovine serum albumin (BSA) assembled as monomolecular layer between source and drain electrodes of a vertical, 4-nm-gap hybrid transistor, a marked gate potential dependence as well as sizable currents have been obtained (Mentovich et al., 2009). BSA has also been used as an electronic material after proper doping with C60 molecules (Mentovich et al., 2012), highlighting once more the use of a protein matrix as a promising material for (self)assembling electronic devices. As already stated, among the proteins that have been considered for bioelectronics purposes, a peculiar role is that played by redox metalloproteins, i.e., proteins containing one or more metal ions in their active centers that physiologically accomplish the task of transferring electrons between molecular partners. Metalloproteins represent about 30% of the entire proteome and the subset of the redox ones, including blue copper proteins, hemebased proteins, iron-sulfur complexes, molybdenum enzymes, and chlorophyll-bearing proteins, are specifically devoted to, or involve in their functioning, electron transfer. Indeed, all of these classes have been considered in bioelectronics applications. Among the most relevant attempts to involve redox metalloproteins in solid-state electronic devices, it is worth recalling implementations exploiting the blue copper metalloprotein azurin, which will be the subject of several studies also reported in Chapter 4.
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Dealing with electronic transport properties studied at the level of a single or a few proteins, scanning probe microscopy-related techniques provide a useful experimental setting. In sections 4.5 and 4.5.1 we will concentrate on the particular technique of electrochemical scanning tunneling microscopy. Here, it is worth recalling that some studies involving single/few molecules by means of current probe atomic force microscopy (CPAFM) on the blue copper protein azurin (see section 4.1.1) have revealed an applied load dependence in the I-V curves, enabling a separation of the effects of distance and tunneling barrier variation in current changes upon increasing load (Zhao et al., 2004). Furthermore, load and bias voltagedependent negative differential resistance (NDR) features suggested a role for the active site redox ion in mediating electron transport through the molecule (Davis et al., 2006, 2008).
1.2 Proteins and planar devices The data retrieved from surface-immobilized redox metalloprotein samples by scanning probe techniques operated in the electrochemical environment (see Chapter 4) suggest clearly that these molecules behave like molecular switches, being able to allow or prevent electrons flowing through them according to the availability of molecular electronic levels in between the Fermi levels of tip and substrate, considering also the coupling of these levels with the environment. The possibility of switching the conduction state of an object such as a molecule by gating the flow of electrons through it represents indeed the basic feature of a molecular (nano)transistor (see Figure 4.21). Therefore, the idea of trying to implement a single (few) molecule(s) transistor exploiting the electron conduction properties of metalloproteins (e.g., azurin) arises naturally (Facci, 2002). Redox metalloproteins arranged in one-molecule-thick films in a gap defined by a pair of planar electrodes could hence act as a channel in a field-effect three-terminal device. Of course, the problems involved in implementing such a device immediately appear to be very serious and numerous. Among them, it is worth stressing two of the most relevant ones, which are: (i) the implementation and theoretical description of effective electrical contacts between metalloproteins and metal electrodes, and (ii) the understanding and optimization of the mechanisms and conditions of intermolecular electron transfer in 2D ensembles of metalloproteins, considering also the fact that many of these devices are implemented in water-less conditions (only tightly bound water molecules are present). Here, we will not discuss
1.2 Proteins and planar devices
in detail the necessary conditions for the construction of these devices, i.e., the strategies for protein immobilization on a surface. We refer the reader interested in this topic to reviews in the literature (Willner & Katz, 2000; Lo¨sche, 1997; Ulman, 2001). Albeit a final solution for these classes of problems is still lacking, the practical realization of working devices is possible and it has been accomplished in fact. Indeed, it has been shown that proteins in a dry environment have an electronic conductivity comparable to that of conjugated molecules, highlighting the fact that proteins offer a very efficient medium for electron transport (see section 4.4.1). It is typically assumed that electron transport between two electrodes separated by a monomolecular protein layer has an exponential dependence on the tunneling barrier width (the thickness of the protein layer can be used as a good approximation for the barrier width). The decay constant can be exploited to investigate the type of transport mechanism, specifically to distinguish between a superexchange or a hopping mechanism (Ron et al., 2010). In fact, it is in principle possible to distinguish between a direct non-resonant tunneling and a multistep tunneling process (Ron et al., 2010). It is also possible that the characterization of these devices could provide information on the basic transfer properties of proteins even if they are studied in an environment different from that in which they perform their natural task. The implementation of a Field Effect Transistor (FET) e or “single particle transistor-like protein device” e should take advantage of state-of-the-art lithographic techniques for the definition of planar (nano)electrodes. Figure 4.21 depicts the operating principle of a generic single metalloprotein planar transistor. Here, a redox metalloprotein is located in the nanometersized gap between two planar electrodes and is electrostatically coupled to a gate electrode. The coupling is responsible for shifting the electronic levels of the molecule with respect to the Fermi levels of the metal leads, enabling or hindering the electronic flow via the aforementioned levels. In this case, the choice of using metalloproteins is dictated by the possibility of exploiting energy levels that are specific to redox molecules. The idea is to use the typical features observed in electrochemical experiments on these molecules (see section 4.4.1) to perform a task in the context of solid-state electronics. To date, a limited number of different planar nanoelectronic devices have been implemented. They typically make use of those redox metalloproteins, which have so far shown the most robust performance in withstanding large electric fields without undergoing denaturation and in surviving rather non physiological environments such as that of the surface of a hybrid electronic device (operated in a water-less environment).
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The implementations reported so far have been for two- and threeterminal devices; they have had the aim of demonstrating the feasibility of such hybrid devices and have been used especially to characterize those particular systems, trying to shed light on the role of molecular organization and on that of the particular metal ions in the protein. The first implementation of a hybrid nanoelectronic device was of a solidstate molecular rectifier based on a (sub)monolayer of azurin molecules (Rinaldi et al., 2002). In this case, the main interest was in the transport properties of the protein layer. A monomolecular carpet of metalloproteins was chemisorbed on the surface of thermally grown SiO2 in between two gold nanoelectrodes (Cr-Au) defined by e-beam lithography (EBL). The chemistry used for the chemisorption of the protein carpet was based on silanes. The construct guaranteed a covalent immobilization of the molecules on the surface as well as a defined uniform protein orientation. Control experiments on non-uniformly oriented azurin hybrid devices were performed using another immobilization strategy. The achievement of a uniformly oriented protein film in between nanoelectrodes is of extreme importance for the performance of a hybrid device for two main reasons: (i) it enables a higher surface coverage, which can facilitate intermolecular electron transfer between neighboring metalloproteins; (ii) for a given protein coverage, it endows the molecular carpet with a uniformity in the location and orientation of the Cu-containing redox active sites, increasing the probability of electron transfer. Another important aspect, which is strictly related to protein orientation at the surface, is protein electrostatics. Proteins, being complex zwitterionic molecules, possess on their surface a variety of charged and polar groups whose global effect is described by MEPs (molecular electrostatic potentials). MEPs are recognized to play a major role in protein interaction and bio-recognition in solution and drive the phenomena of self-assembling at surfaces and adsorption kinetics. MEPs for azurin have been computed in a range of different conditions involving different protein oxidation states, solution pH and ionic strength. Calculations have shown that azurin, in the experimental conditions used in the work being discussed, possesses a strong dipolar moment (150 D) that can sum up in an oriented, 2D molecular assembly, influencing the overall field sensed by electrons flowing in the monomolecular layer. The electrical characterization of a two-electrode device with a 60 nm gap has been performed in the case of uniformly versus randomly oriented protein immobilization. Some important features and differences were found: (i) the uniformly oriented sample showed a marked rectifying behavior (rectification ratio of 175 at 1.5 V vs a ratio of just 10 for the randomly oriented
1.2 Proteins and planar devices
counterpart); (ii) the uniformly oriented sample showed a 10-fold higher current with respect to the randomly oriented one; this effect, along with that of rectification, can be attributed to the molecular dipoles summing-up in the self-assembled edifice and providing an electrostatic field superimposing on the external bias; (iii) marked steps in the I-V curves, tentatively ascribable to the involvement in the conduction mechanism of protein redox levels, appeared in both samples, albeit different in intensity. This first demonstration of a metalloprotein-based molecular rectifier showed poor stability to aging and to environmental conditions, suggesting, however, that the nature of the observed phenomena was due to the presence of the molecules. Another work addressed several open issues related to protein sample purity, and the role of the Cu ion in the azurin active site (Rinaldi et al., 2003). In nanogaps 50 to 100 nm wide, it turned out that recombinant native azurin performed better that the wild type since it showed a rectifying ratio of 500 at 10 V. Moreover, an extensive study with different molecules (recombinant native, Zn-azurin, and apo-azurin e without the metal ion), all immobilized in a uniform orientation, was undertaken in order to understand the role of the particular metal ion in the active site, in a similar fashion to what was previously studied by EC-STM at the level of the single molecule (Facci et al., 2001; Alessandrini et al., 2003). These results confirmed the key role of Cu ions and that of the electronic levels brought about by ion presence, in assisting current flow through the molecular carpet. Zn-azurin and apo-azurin, albeit almost identical to native Cu-azurin in molecular structure, were in fact unable to let any appreciable current flow in the device. Finally, the role of relative humidity in preserving the performance of the hybrid device over time was revealed. A relative humidity of around 50% was found to be optimal for device performance and durability. As a further step, a three-electrode device has been implemented (Maruccio et al., 2005), i.e., a device where the current through the molecular carpet was controlled by a gate electrode in a FET-like implementation. In this particular case, attention was devoted to the possibility of finding in the electron transport phenomenon features that could resemble a transistor-like effect identified in EC-STM experiments (see section 4.6) and related to the redox properties of the proteins. The experimental configuration for such a device was pretty much the same as the two-electrode one (EBL defined Cr/Au arrow-shaped nanoelectrodes facing a 100 nm gap on a Si/SiO2 substrate). However, the main difference was the presence of an Ag electrode on the back of the structure, making an ohmic contact with the p-doped Si substrate. This electrode was used as gate in a FET configuration. The main findings of this work were the presence of a marked resonance in
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the measured transcharacteristics, which displayed a peak-to-valley ratio of 2 at Vg¼ 1.25 V. Only devices assembled from Cu-azurin displayed such a feature, at variance with Zn- and apo-azurin. Interestingly, the switching behavior of this protein-based FET was also modeled in a simplified frame of protein chains of alternating reduced and oxidized molecules, invoking a hopping mechanism for intermolecular electron transfer. Within this framework, it was possible to account for the main experimental features of the implemented device, namely: (i) the appearance of the described resonance in transcharacteristics, and (ii) the marked onset in the I-V curves, as already described in the implementation of the twoterminal devices. Recently, Richter et al. developed a vertical transistor configuration based on a small channel in which proteins were hosted (Mentovich et al., 2009). In this configuration, a central gate electrode was exploited to activate a self-assembled monolayer of proteins sandwiched between two metal electrodes representing the drain and source electrodes. It is to be stressed that, in contrast to the previously described planar devices, in this configuration the current flow was in the same direction as the main axis of the self-assembled proteins, whereas in the previous case the current was perpendicular to the main axes and, moreover, in the set-up by Richter et al., the current was measured over a very small monolayer area. As a consequence, in the former set-up the intermolecular electron transport mechanism had a relevance, which was not present in the latter one, where the main transport occurs across just one protein. It has been shown that the gate potential is able to align the energy levels of the proteins inside the window defined by the Fermi levels of the source and the drain. By this technique it has been possible to ascertain the connection between the redox properties of the proteins and the presence of a negative differential resistance feature in the I-V curves (Mentovich et al., 2011). Once again, the azurin protein was used to perform this experiment and the results have been compared to the case of the apo-azurin protein. The absence of the NDR peak in the case of apo-azurin suggests that the observed feature is related to the redox center of azurin.
1.3 The future of biomolecular electronics After more than two decades of intense activity in the field, it is time to ask ourselves whether this charming discipline is likely to have a future as an effective competitor for conventional electronics or whether its achievements,
1.3 The future of biomolecular electronics
albeit interesting, are to be considered just proofs of principle and will never get to the level required for representing a real alternative to the current technology. This is of course a matter of debate, and it is a waste of time to try to elaborate an answer that is largely shared among the scientist active in the field. Rather, it is perhaps more useful to present some considerations that can contribute to the development of one’s own judgment on the subject. The possibility that a novel technology, relying on approaches so different from those of the current solid-state-electronics-based one, has any chance of replacing or even only supplementing the existing one appears indeed quite unlikely. Several reasons seem to suggest this conclusion; probably the main one is that in such a technologically-intensive field as the modern electronic industry, even the tiniest modification of the production processes requires such a huge economic effort that it is usually unbearable by single companies. In light of this fact, a radical change of perspective like that required by biomolecular electronics (which, for instance, would need to change the fabrication approach from top-down to bottom-up) would represent a genuine revolution, incompatible with the natural development of the market. Even the often-mentioned argument that optical lithography is rapidly approaching its physical limit dictated by the laws of diffraction applied to a radiation of finite wavelength, and that the size of the tiniest features could not decrease further, is regularly falsified by technological advances (to date, the tiniest transistor in production, belonging to the family “Ivy Bridge”, has a size of 22 nm). The recent achievement of carbon nanotube-based transistors, whose production is compatible with the technological infrastructure used for producing CMOS commercial devices, provides a much more reliable insight into the likely near future development of electronics technology (Park et al., 2012). Indeed, the bottom-up approach required for self-assembling biological macromolecules, despite being fascinating, does not provide the reliability required for massive production of identical devices. Using it for device production would require a different capability for rejecting defective pathways in integrated circuits, possibly operating in a dynamic way, thus recalling the behavior of real neural networks, which is, unfortunately, still a long way off for human technology. A further major problem is then represented by the requirement for a water-based environment for a proper functioning of biomolecules (see section 3.5). This difficulty sounds to date almost unsurmountable, in light of the drastic incompatibility that both fabrication processes and device operation have with a wet environment!
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Another obvious concern is connected to the robustness and stability (time, temperature, environmental changes, etc.) of a chip made of biomolecular devices. Even if there exists evidence for the increased thermal stability of biomolecules operated as part of an electronic circuit and shelf life can be made longer than that of the corresponding biomolecules in vivo or freely diffusing in solution, the comparison with solid-state inorganic devices appears frankly unbearable. The current levels sustained by biomolecular devices are another important cause of concern. It is impossible to foresee the implementation of any biomolecular device operating at high power. Last but not least, the best performance dealing with switching ratios obtained with biomolecular electronic devices is by far inferior to that of any, even modest, solid-state device. Perhaps, the most promising and realistic possible application niche where biomolecular devices can strive to compete with solid-state counterparts is that of (bio)sensing. Indeed, the richness in surface chemistry and the intrinsic potential of many biomolecules to undergo conformational changes upon environmental parameter variation and/or binding of molecular effectors make them ideally suited for sensing even the slightest changes, when arranged in single/few-molecule devices as is the case in a bioFET (see for example, section 4.6). It is conceivable that such devices would be integrated with conventional solid-state electronics, providing just a compatible signal for further standard processing and elaboration. At any rate, the general outcome of all these considerations suggests a remarkable reshaping of the extent and potentials of biomolecular electronic devices. Does it mean that there is no future for interfacing biomolecular systems with man-made technology? We believe that the answer to this question is no. Let us see why.
1.4 A novel idea: electrical control of biomolecular systems In most cases, extensive efforts in any field of science and technology bring about relevant byproducts that are largely independent of the success of the original enterprise. It is not uncommon that relevant discoveries and applications come about from research aiming at totally different results (see, for instance, the biography of Irvin Langmuir reported in Tanford, 2004). The awareness developed during more than two decades of research in the field of biomolecular electronics puts researchers active in the field in the position
1.4 A novel idea: electrical control of biomolecular systems
of being able to master many aspects related to interfacing electronic systems and biological ones. Examples include the interplay between surface functionalization and molecular biology, which allows researchers to immobilize a given biomolecule in a desired site with a chosen orientation (Alessandrini et al., 2008); the availability of powerful, single molecule investigation and manipulation techniques such as scanning probe microscopes (Alessandrini & Facci, 2005); the deep understanding of the role of contacts with electrodes in measuring the electrical properties of single molecules; the role of solid-state surfaces in affecting biomolecular conformation and stability, just to quote some of the most relevant aspects. On top of these, there is a further fundamental aspect that can be easily recognized as being at the basis of biomolecular electronics: that is the role that electrical phenomena play in biological systems. This is of course a major issue, chemical-physical in nature, that involves nearly all biological phenomena. It is enough to think of the role that electrical interactions play in maintaining the various molecular components of a biological system in solution, preventing their flocculation, the otherwise unavoidable fate of any colloidal system subjected only to Van der Waals-type forces (Israelachvili, 2005). This pervasive role of electrostatic interactions in biological systems is connected to the properties of the chemical moieties forming the various biopolymers, which are often zwitterionic or charged (Branden & Tooze, 1999), as well as to the presence of a polar solvent that dissociates salts dissolved in it. The role of electrical interaction, however, affects a very large number of biological phenomena in a much more detailed and refined way. Just to quote some examples, we can think of the role of ions such as Naþ and Kþ in generating and maintaining transmembrane potential across the plasma membrane; the effect of charged residues on the conformation and conformational modifications of enzymes, antibodies, and many other proteins; the peculiar, albeit fundamental, function that positively charged residues (e.g., arginines) have in enabling the response of voltage-gated ion channels to changes in transmembrane potential (see section 3.6.1); the major role that biological redox reactions play in nearly all the key metabolic, energetic, and control phenomena in cells; the effect that modulation of surface charges has in controlling cell aggregation (e.g., human erythrocytes possess a net negative charge, y 2.5$1012 C, on their surface that prevents them from clumping together; treating cells with a proteolytic enzyme that cleaves the charged residues on their surface proteins causes red blood cell clotting).
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At the systemic level too, biological matter is governed by electrical interactions and phenomena and relevant examples are really countless. Just considering the activity of neurons and the nervous system in general, or the behavior of cardiomyocytes or muscles, one has a glimpse into the pervasive role that these phenomena play. Of course, such a major role of electrostatic interaction in nearly all the structural and functional aspects of biology is also routinely exploited by technology in many contexts. The charges brought about by biological macromolecules, for instance, are used to separate biomolecules according to their mobility in an electric field when one performs gel electrophoresis. Biologists take advantage of electric charges for sorting cells in flow cytometers, and doctors apply electric shocks across patients’ chests in the case of heart attacks or arrhythmias to reestablish the normal rhythm. Whereas there is no point in trying to list all the relevant cases in which electrical interactions play a role when considering biological matter and phenomena, it is instead very important to reflect on the potential relationship that exists between these phenomena and our ability to control them. This is really a point of key relevance for this book, since all the rest of the present work will stem from the considerations we will develop here. Indeed, on the one hand we are now quite convinced of the relevance and ubiquity of electrical phenomena in biological matter (the most sophisticated one in the known Universe) and systems. On the other hand, we are constantly exposed to the most advanced technology that humankind has ever developed, which is electronics. From the research activity in biomolecular electronics we have learnt a lot about how to handle biological entities and how to interface them with the electronic environment. It is therefore possible to make a further step towards the synthesis of these two realms, biology and electronics, to come to a new level of interaction between them. In other words we are about to enter the charming field of extending technological (electrical) control over biological phenomena and systems. This control, as will be thoroughly exposed in the following chapters, will exploit the peculiar features characterizing the handling of electric fields in a water-based salty environment, referring therefore to the concepts of electrochemistry, and will be extended to treating phenomena that, so far, have not been considered amenable to direct electrical control, such as the modulation of gene expression level in bacteria (see Chapter 7) or the extraction of energy from the photosynthetic cycle in plants (see Chapter 8). This novel concept aims at identifying those biological systems that, more than others, appear to be amenable to a seamless integration with electronics
1.5 References
(e.g., power supplies, amplifiers, potentiostats, etc.) and have the potential to give rise to hybrid systems where technological control of a particular aspect such as the oxidation state of certain molecules can drive and rule the behavior of a biological system in a desired direction. We will have the opportunity to realize how large is the extent of phenomena that can in principle be subjected to external electrical control, ranging far beyond the simplest paradigmatic examples of photosynthetic bacteria and organelles and involving, for example, stem cell differentiation, modulation of tumor suppressor (e.g., p53) activity, and electrical blood pressure control (see chapter 8). In the next chapters the reader will be able to appreciate how the heritage left by biomolecular electronics in many relevant scientific and technological contexts can really boost the novel field presented here towards a future that we foresee as bright and rich in potentially charming consequences.
1.5 References Alessandrini, A., & Facci, P. (2005). AFM: a versatile tool in biophysics. Meas. Sci. Technol., 16, R65eR92. Alessandrini, A., Gerunda, M., Canters, G. W., Verbeet, M. P., & Facci, P. (2003). Electron tunnelling through azurin is mediated by the active site Cu ion. Chem. Phys. Lett., 376, 625e630. Alessandrini, A., Berti, L., Gazzadi, G. C., & Facci, P. (2008). Imparting chemical specificity to nanometer-spaced electrodes. Nanotechnology, 19(355303), 1e5. Aviram, A., & Ratner, M. A. (1974). Molecular rectifiers. Chem. Phys. Lett., 29, 277e283. Ban, G., Dong, R., Li, K., Han, H., & Yan, H. (2009). Study on the electric conductivity of Ag-doped DNA in transverse direction. Nanoscale Res. Lett., 4, 321e326. Bernards, D. A., Malliaras, G. G., Toombes, G. E. S., & Gruner, S. M. (2006). Gating of an organic transistor through a bilayer lipid membrane with ion channels. Appl. Phys. Lett., 89(053505), 1e3. Berti, L., Alessandrini, A., & Facci, P. (2005). DNA-templated photoinduced silver deposition. J. Am. Chem. Soc., 127, 11216e11217. Bezanilla, F. (2005). Voltage-gated ion channels. IEEE Trans. Nanobiosci., 4, 34e48. Branden, C., & Tooze, J. (1999). Introduction to protein structure (2nd ed.). New York: Garland. Chen, Y. S., Hong, M. Y., & Huang, G. S. (2012). A protein transistor made of an antibody molecule and two gold nanoparticles. Nat. Nanotechnol., 7, 197e203.
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Davis, J. J., Wang, N., Morgan, A., Zhang, T., & Zhao, J. (2006). Metalloprotein tunnel junctions: compressional modulation of barrier height and transport mechanism. Faraday Discuss., 131, 167e179. Davis, J. J., Peters, B., & Xi, W. (2008). Force modulation and electrochemical gating of conductance in cytochrome. J. Phys.: Condens. Matter, 20(374123), 1e9. Facci, P. (2002). Single metalloproteins at work: towards a single-protein transistor. In T. Chakraborty, F. Peeters, & U. Sivan (Eds.), Nano-physics & bio-electronics: A new Odyssey. Amsterdam: Elsevier. Facci, P., Alliata, D., & Cannistraro, S. (2001). Potential-induced resonant tunneling through a redox metalloprotein investigated by electrochemical scanning probe microscopy. Ultamicroscopy, 89, 291e298. Fodor, J., & Piattelli-Palmarini, M. (2010). What Darwin got wrong (1st ed.). New York: Farrar, Strauss and Giroux. Guo, X., Gorodetsky, A. A., Hone, J., Barton, J. K., & Nuckolls, C. (2008). Conductivity of a single DNA duplex bridging a carbon nanotube gap. Nat. Nanotechnol., 3, 163e167. Huang, S. C., Artyukhin, A. B., Misra, N., Martinez, J. A., Stroeve, P. A., Grigoropoulos, C. P., Ju, J. W., & Noy, A. (2010). Carbon nanotube transistor controlled by a biological ion pump gate. Nano Lett., 10, 1812e1816. Israelachvili, J. N. (2005). Intermolecular and surface forces (3rd ed.). New York: Academic Press. Lo¨sche, M. (1997). Protein monolayers at interfaces. Curr. Opin. Solid State Mater. Sci., 2, 546e556. Maruccio, G., Biasco, A., Visconti, P., Bramanti, A., Pompa, P. P., Calabi, F., Cingolani, R., Rinaldi, R., Corni, S., Di Felice, R., Molinari, E., Verbeet, M. P., & Canters, G. W. (2005). Towards protein field-effect transistors: report and model of a prototype. Adv. Mater., 17, 816e822. Mentovich, E. D., Belgorodsky, B., Kalifa, I., Cohen, H., & Richter, S. (2009). Large-scale fabrication of 4-nm-channel vertical protein-based ambipolar transistors. Nano Lett., 9, 1296e1300. Mentovich, E. D., Belgorodsky, B., & Richter, S. (2011). Resolving the mystery of the elusive peak: negative differential resistance in redox proteins. J. Phys. Chem. Lett., 2, 1125e1128. Mentovich, E., Belgorodsky, B., Gozin, M., Richter, S., & Cohen, H. (2012). Doped biomolecules in miniaturized electric junctions. J. Am. Chem. Soc., 134, 8468e8473. Misra, N., Martinez, J. A., Huang, S.-C. J., Wang, Y., Stroeve, P., Grigoropoulos, C. P., & Noy, A. (2009). Bioelectronic silicon nanowire devices using functional membrane proteins. Proc. Natl. Acad. Sci. USA, 106, 13780e13784. Park, H., Afzali, A., Han, S.-J., Tulevski, G. S., Franklin, A. D., Tersoff, J., Hannon, J. B., & Haensch, W. (2012). High-density integration of carbon nanotubes via chemical self-assembly. Nat. Nanotechnol., 7, 787e791.
1.5 References
Rinaldi, R., Biasco, A., Maruccio, G., Cingolani, R., Alliata, D., Andolfi, L., Facci, P., De Rienzo, F., Di Felice, R., & Molinari, E. (2002). Solid-state molecular rectifier based on self-organized metalloproteins. Adv. Mater., 14, 1449e1453. Rinaldi, R., Biasco, A., Maruccio, G., Cingolani, R., Alliata, D., Andolfi, L., Facci, P., De Rienzo, F., Di Felice, R., Molinari, E., Verbeet, M., & Canters, G. (2003). Electronic rectification in protein devices. Appl. Phys. Lett., 82, 472e474. Ron, I., Sepunaru, L., Itzhakov, S., Belenkova, T., Friedman, N., Pecht, I., Sheves, M., & Cahen, D. (2010). Proteins as electronic materials: electron transport through solid-state protein monolayer junctions. J. Am. Chem. Soc., 132, 4131e4140. Tanford, C. (2004). Ben Franklin stilled the waves. Oxford: Oxford University Press. Ulman, A. (2001). Formation and structure of self-assembled monolayers. Chem. Rev., 96, 1533e1554. Willner, I., & Katz, E. (2000). Integration of layered redox proteins and conductive supports for bioelectronic applications. Angew. Chem. Int. Ed., 39, 1180e1218. Zhao, J. W., Davis, J. J., Sansom, M. S. P., & Hung, A. (2004). Exploring the electronic and mechanical properties of protein using conducting atomic force microscopy. J. Am. Chem. Soc., 126, 5601e5069.
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Biomolecular Electronics
1
CHAPTER OUTLINE 1.0 What is biomolecular electronics? ...........................................................1 1.1 Proteins and biomolecular electronics......................................................3 1.2 Proteins and planar devices.....................................................................6 1.3 The future of biomolecular electronics ...................................................10 1.4 A novel idea: electrical control of biomolecular systems .........................12 1.5 References ............................................................................................15
1.0 What is biomolecular electronics? With the expression “biomolecular electronics” we refer nowadays to that branch of technology that exploits molecules or molecular systems of biological origin for interaction with modern electronics. The idea of using biomolecules to assemble hybrid electronic devices stems from molecular electronics (Aviram & Ratner, 1974) and, as such, is intimately connected with the advent of nanosciences and nanotechnologies, dating back to the beginning of the last decade of the past century. The interest in biomolecules stems not only from their size, which is typically in the nanometer range (at least in two spatial dimensions). Rather, it is often connected to a particular standpoint from which one can regard them. Indeed, an interesting point of view is that of regarding biomolecules in general and proteins in particular as self-contained, nanometer-sized functional units that are highly specialized and efficient in performing a certain functional task. Their efficiency in performing a particular activity is traced back to the fact that biomolecules, being parts of living beings, have naturally evolved over billions of years, reaching the current “optimal” degree of specialization for accomplishing a given task. One can often come across this opinion reading the specialized literature or attending topical conferences; however, it appears quite questionable in light of a slightly less naı¨ve understanding of the Theory of Evolution (Fodor & Piattelli-Palmarini, 2010); nevertheless, it is perhaps a good enough starting point to understand the historical motivations which led to the Biomolecular Electronics. http://dx.doi.org/10.1016/B978-1-4557-3142-8.00001-7 Copyright © 2014 Elsevier Inc. All rights reserved.
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remarkable interest shown by a sizable part of the interdisciplinary scientific community in the use of biomolecules for assembling electronic devices. Another element motivating the interest in proteins as components of electronic circuits is their sizes. Indeed, these molecules often configure self-consistent, functional units with typical sizes in the nanometer range. Therefore, it is conceivable to imagine functional devices as small as a single molecule, especially in view of the fantastic progresses in miniaturization and parallelization made with nano-lithographic techniques. Furthermore, the rich chemistry that particularly characterizes proteinforming monomers, i.e., the 20 natural amino acids, makes these molecules amenable to various kinds of chemical functionalization, thus improving their reactivity towards desired functional groups and enabling, as a consequence, their ready chemisorption on pre-functionalized structures and surfaces. Moreover, the exceptionally fine-tuned recognition properties of biological structures (e.g., complementary nucleic acid strands, DNA-binding proteins, antibody-antigen or receptor-ligand pairs, etc.) could allow for selfassembling of more complex structures in the nanometer-sized gap between meso/nanoscale electrodes. Among the various classes of molecules of biological origin, two of them have been especially focused upon by researchers in the field: DNA and proteins. The reasons for this choice are different; in the case of DNA, its robustness and ease of handling are relevant factors. Nevertheless, a further important aspect fostering DNA as an interesting molecule in biomolecular electronics stemmed from the idea that, if we could understand the mechanism by which electrons are driven along the double helix during DNA repair of damage caused by oxidative stress, we would obtain, as a byproduct, a way to produce molecular wires, that is, one-moleculethick conducting wires, to be used in assembling (bio)molecular circuitry. If DNA can be regarded in terms of molecular wire, what else does one need to assemble molecular circuits? Some functionally relevant electronic elements, of course. Those were identified as the proteins, which are indeed deputed to perform the main functional tasks in the organisms that express them. At this point a relevant question arose that has not been completely answered yet, about the electrical conductivity of biomolecules. It is indeed obvious that at the basis of any electronic circuit there is a need to deal with wires and elements that can conduct or, more generally, interact functionally with electron flows, i.e., electron currents. Whereas this issue is still open in the case of DNA, which displays good conductivity over short ranges (of the order of nanometers) and appears to be
1.1 Proteins and biomolecular electronics
an insulator over larger distances (Guo et al., 2008), the situation regarding proteins is more complex and requires detailed analysis because of the large variability of their properties (mirroring the differences in protein structures). In the case of DNA, its poor conductivity, at least in the mesoscopic range, has prompted various approaches for enhancing it, based on doping (with metal ions or intercalating agents (Ban et al., 2009)) or on the use of DNA as a molecular template for metal nanowire growth (Berti et al., 2005). In the case of proteins, which possess an intrinsically higher chemical and structural variability compared with DNA, it is not surprising that specific transport proteins, carrying either electrons or ions, soon attracted the interest of researchers.
1.1 Proteins and biomolecular electronics Typically, electron transport proteins are water-soluble molecules (rare exceptions apart; see, e.g., cytochrome b561, a transmembrane three-heme cytochrome), whereas ion transport proteins are membrane molecules requiring specific environmental conditions (incorporation in a lipid bilayer) to be functionally active (see section 3.6.1). According to this difference, whereas the former are more easily implemented in electronic circuits, the latter are more demanding to exploit in a nano-circuit configuration. Ion channels have been used to gate transistors in hybrid devices thanks to their selectivity for specific ions (Bernards et al., 2006) and the inherent similarity of the gating mechanism of voltage-gated ion channels to the basic features of a field-effect transistor (Bezanilla, 2005). A specific application of ion channels in electronic circuits for sensing applications is the coupling of the channels reconstituted in lipid bilayers with other electronic nanostructures such as semiconductor nanowires (Misra et al., 2009). Examples of these applications are the possibility, by changing the pH of the solution, of controlling the conductance of a silicon nanowire covered with a lipid bilayer integrating peptide pores and the creation of nanobioelectronic transistors exploiting ion pumps such as Naþ/Kþ-ATPase (Huang et al., 2010). However, the most popular and charming class of proteins that seemed to be particularly suitable for implementing electronic devices is the redox metalloproteins. These very important molecules are indeed able to shuttle electrons between molecular partners by reversibly changing the oxidation state of the metal ions they contain (see sections 4.1, 4.1.1, and 4.1.2). Following the route defined by conventional solid-state electronics, the research efforts in biomolecular electronics have generally focused on
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demonstrating transistor-like behavior that proteins might display when positioned between two electrodes. However, considering the classic definition of a transistor, we should stress here a different meaning for the same word. A transistor represents a device composed of at least three electrodes (source, drain, and gate) which is able to amplify or switch the current between a pair of electrodes (e.g., source-drain) by controlling the voltage or current between two of these three electrodes (e.g., source-gate or drain-gate). In conventional electronics the controlling signal is an electric signal. In the context of proteins, among the possible signals that can function for gating a hybrid device we include also other signals of a chemical or physical nature. Among them one has to consider all the parameters that are able to modify, at constant bias voltage, the transport properties of a protein or of a protein layer sandwiched between two electrodes. In a similar way, protein conformational variations can induce changes in its transport properties or in those of another nanostructure to which the protein is coupled. Among these signals one can list variation in environmental pH, temperature, substrate recognition by enzymes and so on. The sensitivity to any of these signals confers sensing properties to the related devices. We will consider the main results obtained along the pathway of understanding the behavior of redox metalloproteins when used as active parts of hybrid electronic devices in Chapter 4. Here, we continue by noting that other features of proteins have also attracted researchers’ attention and have been used in various attempts to exploit the properties of natural biomolecules to implement electronic devices. There is, for instance, the case of antibodies. These molecules are part of an organism’s immune system and are known for their specific recognition and binding capabilities against the corresponding antigens. Their use has been recently demonstrated in assembling single-protein devices (Chen et al., 2012). Particularly, antibodies of the IgG type (immunogammaglobulin) have been raised against Au nanoparticles, 5 nm in diameter. An IgG molecule can bind up to two of these gold nanoparticles, one per Fab fragment, where the specific recognition sites for antigens are located. This specific binding provides a stable bond. Gold nanoparticles are attached to e-beam lithography-fabricated 10-nm gap electrodes that act as source and drain electrodes. Once the electrodes have been decorated with nanoparticles, they are exposed to IgGs that specifically recognize the nanoparticles, binding them firmly. This results in a configuration that can be gated electrically, by means of a proximal electrode, and optically, by the action of a CdSe Qdot attached to the IgG molecule at its Fc fragment. While irradiated, the Qdot absorbs light, providing an induced dipole moment
1.1 Proteins and biomolecular electronics
(confined exciton) that operates an effective gating of the single-protein transistor, probably mediated by conformational modifications occurring in the biomolecule. A richness of features is observed, including current values of the order of a few hundreds of picoamps, rectification, and negative differential resistance (NDR), which is ascribed to the presence of the Fc fragment. Such an exciting implementation is surprising because it shows it is possible for an electric current to flow through a large biomolecule that, physiologically, is not thought to have any electron transport capabilities. This fact raises questions about protein conductivity and, hence, about the suitability of proteins as materials for electronics. Whereas theoretical considerations about protein conductivity and electron transfer in general will be dealt with later in this book (see Chapter 4), systematic experimental studies (Ron et al., 2010) suggest that electron transport through proteins, even measured in the solid state, is more effective than through a generic insulating material. There, the peculiar conductivity displayed by redox metalloproteins is confirmed, but even proteins without a physiological role in electron transfer (e.g., albumin, bacteriorhodopsin) display electron transport ˚ 1 that denote them as promising decay constants b in the range 0.12e0.27 A materials for bioelectronic applications. Indeed, with the same molecule, bovine serum albumin (BSA) assembled as monomolecular layer between source and drain electrodes of a vertical, 4-nm-gap hybrid transistor, a marked gate potential dependence as well as sizable currents have been obtained (Mentovich et al., 2009). BSA has also been used as an electronic material after proper doping with C60 molecules (Mentovich et al., 2012), highlighting once more the use of a protein matrix as a promising material for (self)assembling electronic devices. As already stated, among the proteins that have been considered for bioelectronics purposes, a peculiar role is that played by redox metalloproteins, i.e., proteins containing one or more metal ions in their active centers that physiologically accomplish the task of transferring electrons between molecular partners. Metalloproteins represent about 30% of the entire proteome and the subset of the redox ones, including blue copper proteins, hemebased proteins, iron-sulfur complexes, molybdenum enzymes, and chlorophyll-bearing proteins, are specifically devoted to, or involve in their functioning, electron transfer. Indeed, all of these classes have been considered in bioelectronics applications. Among the most relevant attempts to involve redox metalloproteins in solid-state electronic devices, it is worth recalling implementations exploiting the blue copper metalloprotein azurin, which will be the subject of several studies also reported in Chapter 4.
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Dealing with electronic transport properties studied at the level of a single or a few proteins, scanning probe microscopy-related techniques provide a useful experimental setting. In sections 4.5 and 4.5.1 we will concentrate on the particular technique of electrochemical scanning tunneling microscopy. Here, it is worth recalling that some studies involving single/few molecules by means of current probe atomic force microscopy (CPAFM) on the blue copper protein azurin (see section 4.1.1) have revealed an applied load dependence in the I-V curves, enabling a separation of the effects of distance and tunneling barrier variation in current changes upon increasing load (Zhao et al., 2004). Furthermore, load and bias voltagedependent negative differential resistance (NDR) features suggested a role for the active site redox ion in mediating electron transport through the molecule (Davis et al., 2006, 2008).
1.2 Proteins and planar devices The data retrieved from surface-immobilized redox metalloprotein samples by scanning probe techniques operated in the electrochemical environment (see Chapter 4) suggest clearly that these molecules behave like molecular switches, being able to allow or prevent electrons flowing through them according to the availability of molecular electronic levels in between the Fermi levels of tip and substrate, considering also the coupling of these levels with the environment. The possibility of switching the conduction state of an object such as a molecule by gating the flow of electrons through it represents indeed the basic feature of a molecular (nano)transistor (see Figure 4.21). Therefore, the idea of trying to implement a single (few) molecule(s) transistor exploiting the electron conduction properties of metalloproteins (e.g., azurin) arises naturally (Facci, 2002). Redox metalloproteins arranged in one-molecule-thick films in a gap defined by a pair of planar electrodes could hence act as a channel in a field-effect three-terminal device. Of course, the problems involved in implementing such a device immediately appear to be very serious and numerous. Among them, it is worth stressing two of the most relevant ones, which are: (i) the implementation and theoretical description of effective electrical contacts between metalloproteins and metal electrodes, and (ii) the understanding and optimization of the mechanisms and conditions of intermolecular electron transfer in 2D ensembles of metalloproteins, considering also the fact that many of these devices are implemented in water-less conditions (only tightly bound water molecules are present). Here, we will not discuss
1.2 Proteins and planar devices
in detail the necessary conditions for the construction of these devices, i.e., the strategies for protein immobilization on a surface. We refer the reader interested in this topic to reviews in the literature (Willner & Katz, 2000; Lo¨sche, 1997; Ulman, 2001). Albeit a final solution for these classes of problems is still lacking, the practical realization of working devices is possible and it has been accomplished in fact. Indeed, it has been shown that proteins in a dry environment have an electronic conductivity comparable to that of conjugated molecules, highlighting the fact that proteins offer a very efficient medium for electron transport (see section 4.4.1). It is typically assumed that electron transport between two electrodes separated by a monomolecular protein layer has an exponential dependence on the tunneling barrier width (the thickness of the protein layer can be used as a good approximation for the barrier width). The decay constant can be exploited to investigate the type of transport mechanism, specifically to distinguish between a superexchange or a hopping mechanism (Ron et al., 2010). In fact, it is in principle possible to distinguish between a direct non-resonant tunneling and a multistep tunneling process (Ron et al., 2010). It is also possible that the characterization of these devices could provide information on the basic transfer properties of proteins even if they are studied in an environment different from that in which they perform their natural task. The implementation of a Field Effect Transistor (FET) e or “single particle transistor-like protein device” e should take advantage of state-of-the-art lithographic techniques for the definition of planar (nano)electrodes. Figure 4.21 depicts the operating principle of a generic single metalloprotein planar transistor. Here, a redox metalloprotein is located in the nanometersized gap between two planar electrodes and is electrostatically coupled to a gate electrode. The coupling is responsible for shifting the electronic levels of the molecule with respect to the Fermi levels of the metal leads, enabling or hindering the electronic flow via the aforementioned levels. In this case, the choice of using metalloproteins is dictated by the possibility of exploiting energy levels that are specific to redox molecules. The idea is to use the typical features observed in electrochemical experiments on these molecules (see section 4.4.1) to perform a task in the context of solid-state electronics. To date, a limited number of different planar nanoelectronic devices have been implemented. They typically make use of those redox metalloproteins, which have so far shown the most robust performance in withstanding large electric fields without undergoing denaturation and in surviving rather non physiological environments such as that of the surface of a hybrid electronic device (operated in a water-less environment).
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The implementations reported so far have been for two- and threeterminal devices; they have had the aim of demonstrating the feasibility of such hybrid devices and have been used especially to characterize those particular systems, trying to shed light on the role of molecular organization and on that of the particular metal ions in the protein. The first implementation of a hybrid nanoelectronic device was of a solidstate molecular rectifier based on a (sub)monolayer of azurin molecules (Rinaldi et al., 2002). In this case, the main interest was in the transport properties of the protein layer. A monomolecular carpet of metalloproteins was chemisorbed on the surface of thermally grown SiO2 in between two gold nanoelectrodes (Cr-Au) defined by e-beam lithography (EBL). The chemistry used for the chemisorption of the protein carpet was based on silanes. The construct guaranteed a covalent immobilization of the molecules on the surface as well as a defined uniform protein orientation. Control experiments on non-uniformly oriented azurin hybrid devices were performed using another immobilization strategy. The achievement of a uniformly oriented protein film in between nanoelectrodes is of extreme importance for the performance of a hybrid device for two main reasons: (i) it enables a higher surface coverage, which can facilitate intermolecular electron transfer between neighboring metalloproteins; (ii) for a given protein coverage, it endows the molecular carpet with a uniformity in the location and orientation of the Cu-containing redox active sites, increasing the probability of electron transfer. Another important aspect, which is strictly related to protein orientation at the surface, is protein electrostatics. Proteins, being complex zwitterionic molecules, possess on their surface a variety of charged and polar groups whose global effect is described by MEPs (molecular electrostatic potentials). MEPs are recognized to play a major role in protein interaction and bio-recognition in solution and drive the phenomena of self-assembling at surfaces and adsorption kinetics. MEPs for azurin have been computed in a range of different conditions involving different protein oxidation states, solution pH and ionic strength. Calculations have shown that azurin, in the experimental conditions used in the work being discussed, possesses a strong dipolar moment (150 D) that can sum up in an oriented, 2D molecular assembly, influencing the overall field sensed by electrons flowing in the monomolecular layer. The electrical characterization of a two-electrode device with a 60 nm gap has been performed in the case of uniformly versus randomly oriented protein immobilization. Some important features and differences were found: (i) the uniformly oriented sample showed a marked rectifying behavior (rectification ratio of 175 at 1.5 V vs a ratio of just 10 for the randomly oriented
1.2 Proteins and planar devices
counterpart); (ii) the uniformly oriented sample showed a 10-fold higher current with respect to the randomly oriented one; this effect, along with that of rectification, can be attributed to the molecular dipoles summing-up in the self-assembled edifice and providing an electrostatic field superimposing on the external bias; (iii) marked steps in the I-V curves, tentatively ascribable to the involvement in the conduction mechanism of protein redox levels, appeared in both samples, albeit different in intensity. This first demonstration of a metalloprotein-based molecular rectifier showed poor stability to aging and to environmental conditions, suggesting, however, that the nature of the observed phenomena was due to the presence of the molecules. Another work addressed several open issues related to protein sample purity, and the role of the Cu ion in the azurin active site (Rinaldi et al., 2003). In nanogaps 50 to 100 nm wide, it turned out that recombinant native azurin performed better that the wild type since it showed a rectifying ratio of 500 at 10 V. Moreover, an extensive study with different molecules (recombinant native, Zn-azurin, and apo-azurin e without the metal ion), all immobilized in a uniform orientation, was undertaken in order to understand the role of the particular metal ion in the active site, in a similar fashion to what was previously studied by EC-STM at the level of the single molecule (Facci et al., 2001; Alessandrini et al., 2003). These results confirmed the key role of Cu ions and that of the electronic levels brought about by ion presence, in assisting current flow through the molecular carpet. Zn-azurin and apo-azurin, albeit almost identical to native Cu-azurin in molecular structure, were in fact unable to let any appreciable current flow in the device. Finally, the role of relative humidity in preserving the performance of the hybrid device over time was revealed. A relative humidity of around 50% was found to be optimal for device performance and durability. As a further step, a three-electrode device has been implemented (Maruccio et al., 2005), i.e., a device where the current through the molecular carpet was controlled by a gate electrode in a FET-like implementation. In this particular case, attention was devoted to the possibility of finding in the electron transport phenomenon features that could resemble a transistor-like effect identified in EC-STM experiments (see section 4.6) and related to the redox properties of the proteins. The experimental configuration for such a device was pretty much the same as the two-electrode one (EBL defined Cr/Au arrow-shaped nanoelectrodes facing a 100 nm gap on a Si/SiO2 substrate). However, the main difference was the presence of an Ag electrode on the back of the structure, making an ohmic contact with the p-doped Si substrate. This electrode was used as gate in a FET configuration. The main findings of this work were the presence of a marked resonance in
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the measured transcharacteristics, which displayed a peak-to-valley ratio of 2 at Vg¼ 1.25 V. Only devices assembled from Cu-azurin displayed such a feature, at variance with Zn- and apo-azurin. Interestingly, the switching behavior of this protein-based FET was also modeled in a simplified frame of protein chains of alternating reduced and oxidized molecules, invoking a hopping mechanism for intermolecular electron transfer. Within this framework, it was possible to account for the main experimental features of the implemented device, namely: (i) the appearance of the described resonance in transcharacteristics, and (ii) the marked onset in the I-V curves, as already described in the implementation of the twoterminal devices. Recently, Richter et al. developed a vertical transistor configuration based on a small channel in which proteins were hosted (Mentovich et al., 2009). In this configuration, a central gate electrode was exploited to activate a self-assembled monolayer of proteins sandwiched between two metal electrodes representing the drain and source electrodes. It is to be stressed that, in contrast to the previously described planar devices, in this configuration the current flow was in the same direction as the main axis of the self-assembled proteins, whereas in the previous case the current was perpendicular to the main axes and, moreover, in the set-up by Richter et al., the current was measured over a very small monolayer area. As a consequence, in the former set-up the intermolecular electron transport mechanism had a relevance, which was not present in the latter one, where the main transport occurs across just one protein. It has been shown that the gate potential is able to align the energy levels of the proteins inside the window defined by the Fermi levels of the source and the drain. By this technique it has been possible to ascertain the connection between the redox properties of the proteins and the presence of a negative differential resistance feature in the I-V curves (Mentovich et al., 2011). Once again, the azurin protein was used to perform this experiment and the results have been compared to the case of the apo-azurin protein. The absence of the NDR peak in the case of apo-azurin suggests that the observed feature is related to the redox center of azurin.
1.3 The future of biomolecular electronics After more than two decades of intense activity in the field, it is time to ask ourselves whether this charming discipline is likely to have a future as an effective competitor for conventional electronics or whether its achievements,
1.3 The future of biomolecular electronics
albeit interesting, are to be considered just proofs of principle and will never get to the level required for representing a real alternative to the current technology. This is of course a matter of debate, and it is a waste of time to try to elaborate an answer that is largely shared among the scientist active in the field. Rather, it is perhaps more useful to present some considerations that can contribute to the development of one’s own judgment on the subject. The possibility that a novel technology, relying on approaches so different from those of the current solid-state-electronics-based one, has any chance of replacing or even only supplementing the existing one appears indeed quite unlikely. Several reasons seem to suggest this conclusion; probably the main one is that in such a technologically-intensive field as the modern electronic industry, even the tiniest modification of the production processes requires such a huge economic effort that it is usually unbearable by single companies. In light of this fact, a radical change of perspective like that required by biomolecular electronics (which, for instance, would need to change the fabrication approach from top-down to bottom-up) would represent a genuine revolution, incompatible with the natural development of the market. Even the often-mentioned argument that optical lithography is rapidly approaching its physical limit dictated by the laws of diffraction applied to a radiation of finite wavelength, and that the size of the tiniest features could not decrease further, is regularly falsified by technological advances (to date, the tiniest transistor in production, belonging to the family “Ivy Bridge”, has a size of 22 nm). The recent achievement of carbon nanotube-based transistors, whose production is compatible with the technological infrastructure used for producing CMOS commercial devices, provides a much more reliable insight into the likely near future development of electronics technology (Park et al., 2012). Indeed, the bottom-up approach required for self-assembling biological macromolecules, despite being fascinating, does not provide the reliability required for massive production of identical devices. Using it for device production would require a different capability for rejecting defective pathways in integrated circuits, possibly operating in a dynamic way, thus recalling the behavior of real neural networks, which is, unfortunately, still a long way off for human technology. A further major problem is then represented by the requirement for a water-based environment for a proper functioning of biomolecules (see section 3.5). This difficulty sounds to date almost unsurmountable, in light of the drastic incompatibility that both fabrication processes and device operation have with a wet environment!
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Another obvious concern is connected to the robustness and stability (time, temperature, environmental changes, etc.) of a chip made of biomolecular devices. Even if there exists evidence for the increased thermal stability of biomolecules operated as part of an electronic circuit and shelf life can be made longer than that of the corresponding biomolecules in vivo or freely diffusing in solution, the comparison with solid-state inorganic devices appears frankly unbearable. The current levels sustained by biomolecular devices are another important cause of concern. It is impossible to foresee the implementation of any biomolecular device operating at high power. Last but not least, the best performance dealing with switching ratios obtained with biomolecular electronic devices is by far inferior to that of any, even modest, solid-state device. Perhaps, the most promising and realistic possible application niche where biomolecular devices can strive to compete with solid-state counterparts is that of (bio)sensing. Indeed, the richness in surface chemistry and the intrinsic potential of many biomolecules to undergo conformational changes upon environmental parameter variation and/or binding of molecular effectors make them ideally suited for sensing even the slightest changes, when arranged in single/few-molecule devices as is the case in a bioFET (see for example, section 4.6). It is conceivable that such devices would be integrated with conventional solid-state electronics, providing just a compatible signal for further standard processing and elaboration. At any rate, the general outcome of all these considerations suggests a remarkable reshaping of the extent and potentials of biomolecular electronic devices. Does it mean that there is no future for interfacing biomolecular systems with man-made technology? We believe that the answer to this question is no. Let us see why.
1.4 A novel idea: electrical control of biomolecular systems In most cases, extensive efforts in any field of science and technology bring about relevant byproducts that are largely independent of the success of the original enterprise. It is not uncommon that relevant discoveries and applications come about from research aiming at totally different results (see, for instance, the biography of Irvin Langmuir reported in Tanford, 2004). The awareness developed during more than two decades of research in the field of biomolecular electronics puts researchers active in the field in the position
1.4 A novel idea: electrical control of biomolecular systems
of being able to master many aspects related to interfacing electronic systems and biological ones. Examples include the interplay between surface functionalization and molecular biology, which allows researchers to immobilize a given biomolecule in a desired site with a chosen orientation (Alessandrini et al., 2008); the availability of powerful, single molecule investigation and manipulation techniques such as scanning probe microscopes (Alessandrini & Facci, 2005); the deep understanding of the role of contacts with electrodes in measuring the electrical properties of single molecules; the role of solid-state surfaces in affecting biomolecular conformation and stability, just to quote some of the most relevant aspects. On top of these, there is a further fundamental aspect that can be easily recognized as being at the basis of biomolecular electronics: that is the role that electrical phenomena play in biological systems. This is of course a major issue, chemical-physical in nature, that involves nearly all biological phenomena. It is enough to think of the role that electrical interactions play in maintaining the various molecular components of a biological system in solution, preventing their flocculation, the otherwise unavoidable fate of any colloidal system subjected only to Van der Waals-type forces (Israelachvili, 2005). This pervasive role of electrostatic interactions in biological systems is connected to the properties of the chemical moieties forming the various biopolymers, which are often zwitterionic or charged (Branden & Tooze, 1999), as well as to the presence of a polar solvent that dissociates salts dissolved in it. The role of electrical interaction, however, affects a very large number of biological phenomena in a much more detailed and refined way. Just to quote some examples, we can think of the role of ions such as Naþ and Kþ in generating and maintaining transmembrane potential across the plasma membrane; the effect of charged residues on the conformation and conformational modifications of enzymes, antibodies, and many other proteins; the peculiar, albeit fundamental, function that positively charged residues (e.g., arginines) have in enabling the response of voltage-gated ion channels to changes in transmembrane potential (see section 3.6.1); the major role that biological redox reactions play in nearly all the key metabolic, energetic, and control phenomena in cells; the effect that modulation of surface charges has in controlling cell aggregation (e.g., human erythrocytes possess a net negative charge, y 2.5$1012 C, on their surface that prevents them from clumping together; treating cells with a proteolytic enzyme that cleaves the charged residues on their surface proteins causes red blood cell clotting).
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At the systemic level too, biological matter is governed by electrical interactions and phenomena and relevant examples are really countless. Just considering the activity of neurons and the nervous system in general, or the behavior of cardiomyocytes or muscles, one has a glimpse into the pervasive role that these phenomena play. Of course, such a major role of electrostatic interaction in nearly all the structural and functional aspects of biology is also routinely exploited by technology in many contexts. The charges brought about by biological macromolecules, for instance, are used to separate biomolecules according to their mobility in an electric field when one performs gel electrophoresis. Biologists take advantage of electric charges for sorting cells in flow cytometers, and doctors apply electric shocks across patients’ chests in the case of heart attacks or arrhythmias to reestablish the normal rhythm. Whereas there is no point in trying to list all the relevant cases in which electrical interactions play a role when considering biological matter and phenomena, it is instead very important to reflect on the potential relationship that exists between these phenomena and our ability to control them. This is really a point of key relevance for this book, since all the rest of the present work will stem from the considerations we will develop here. Indeed, on the one hand we are now quite convinced of the relevance and ubiquity of electrical phenomena in biological matter (the most sophisticated one in the known Universe) and systems. On the other hand, we are constantly exposed to the most advanced technology that humankind has ever developed, which is electronics. From the research activity in biomolecular electronics we have learnt a lot about how to handle biological entities and how to interface them with the electronic environment. It is therefore possible to make a further step towards the synthesis of these two realms, biology and electronics, to come to a new level of interaction between them. In other words we are about to enter the charming field of extending technological (electrical) control over biological phenomena and systems. This control, as will be thoroughly exposed in the following chapters, will exploit the peculiar features characterizing the handling of electric fields in a water-based salty environment, referring therefore to the concepts of electrochemistry, and will be extended to treating phenomena that, so far, have not been considered amenable to direct electrical control, such as the modulation of gene expression level in bacteria (see Chapter 7) or the extraction of energy from the photosynthetic cycle in plants (see Chapter 8). This novel concept aims at identifying those biological systems that, more than others, appear to be amenable to a seamless integration with electronics
1.5 References
(e.g., power supplies, amplifiers, potentiostats, etc.) and have the potential to give rise to hybrid systems where technological control of a particular aspect such as the oxidation state of certain molecules can drive and rule the behavior of a biological system in a desired direction. We will have the opportunity to realize how large is the extent of phenomena that can in principle be subjected to external electrical control, ranging far beyond the simplest paradigmatic examples of photosynthetic bacteria and organelles and involving, for example, stem cell differentiation, modulation of tumor suppressor (e.g., p53) activity, and electrical blood pressure control (see chapter 8). In the next chapters the reader will be able to appreciate how the heritage left by biomolecular electronics in many relevant scientific and technological contexts can really boost the novel field presented here towards a future that we foresee as bright and rich in potentially charming consequences.
1.5 References Alessandrini, A., & Facci, P. (2005). AFM: a versatile tool in biophysics. Meas. Sci. Technol., 16, R65eR92. Alessandrini, A., Gerunda, M., Canters, G. W., Verbeet, M. P., & Facci, P. (2003). Electron tunnelling through azurin is mediated by the active site Cu ion. Chem. Phys. Lett., 376, 625e630. Alessandrini, A., Berti, L., Gazzadi, G. C., & Facci, P. (2008). Imparting chemical specificity to nanometer-spaced electrodes. Nanotechnology, 19(355303), 1e5. Aviram, A., & Ratner, M. A. (1974). Molecular rectifiers. Chem. Phys. Lett., 29, 277e283. Ban, G., Dong, R., Li, K., Han, H., & Yan, H. (2009). Study on the electric conductivity of Ag-doped DNA in transverse direction. Nanoscale Res. Lett., 4, 321e326. Bernards, D. A., Malliaras, G. G., Toombes, G. E. S., & Gruner, S. M. (2006). Gating of an organic transistor through a bilayer lipid membrane with ion channels. Appl. Phys. Lett., 89(053505), 1e3. Berti, L., Alessandrini, A., & Facci, P. (2005). DNA-templated photoinduced silver deposition. J. Am. Chem. Soc., 127, 11216e11217. Bezanilla, F. (2005). Voltage-gated ion channels. IEEE Trans. Nanobiosci., 4, 34e48. Branden, C., & Tooze, J. (1999). Introduction to protein structure (2nd ed.). New York: Garland. Chen, Y. S., Hong, M. Y., & Huang, G. S. (2012). A protein transistor made of an antibody molecule and two gold nanoparticles. Nat. Nanotechnol., 7, 197e203.
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Davis, J. J., Wang, N., Morgan, A., Zhang, T., & Zhao, J. (2006). Metalloprotein tunnel junctions: compressional modulation of barrier height and transport mechanism. Faraday Discuss., 131, 167e179. Davis, J. J., Peters, B., & Xi, W. (2008). Force modulation and electrochemical gating of conductance in cytochrome. J. Phys.: Condens. Matter, 20(374123), 1e9. Facci, P. (2002). Single metalloproteins at work: towards a single-protein transistor. In T. Chakraborty, F. Peeters, & U. Sivan (Eds.), Nano-physics & bio-electronics: A new Odyssey. Amsterdam: Elsevier. Facci, P., Alliata, D., & Cannistraro, S. (2001). Potential-induced resonant tunneling through a redox metalloprotein investigated by electrochemical scanning probe microscopy. Ultamicroscopy, 89, 291e298. Fodor, J., & Piattelli-Palmarini, M. (2010). What Darwin got wrong (1st ed.). New York: Farrar, Strauss and Giroux. Guo, X., Gorodetsky, A. A., Hone, J., Barton, J. K., & Nuckolls, C. (2008). Conductivity of a single DNA duplex bridging a carbon nanotube gap. Nat. Nanotechnol., 3, 163e167. Huang, S. C., Artyukhin, A. B., Misra, N., Martinez, J. A., Stroeve, P. A., Grigoropoulos, C. P., Ju, J. W., & Noy, A. (2010). Carbon nanotube transistor controlled by a biological ion pump gate. Nano Lett., 10, 1812e1816. Israelachvili, J. N. (2005). Intermolecular and surface forces (3rd ed.). New York: Academic Press. Lo¨sche, M. (1997). Protein monolayers at interfaces. Curr. Opin. Solid State Mater. Sci., 2, 546e556. Maruccio, G., Biasco, A., Visconti, P., Bramanti, A., Pompa, P. P., Calabi, F., Cingolani, R., Rinaldi, R., Corni, S., Di Felice, R., Molinari, E., Verbeet, M. P., & Canters, G. W. (2005). Towards protein field-effect transistors: report and model of a prototype. Adv. Mater., 17, 816e822. Mentovich, E. D., Belgorodsky, B., Kalifa, I., Cohen, H., & Richter, S. (2009). Large-scale fabrication of 4-nm-channel vertical protein-based ambipolar transistors. Nano Lett., 9, 1296e1300. Mentovich, E. D., Belgorodsky, B., & Richter, S. (2011). Resolving the mystery of the elusive peak: negative differential resistance in redox proteins. J. Phys. Chem. Lett., 2, 1125e1128. Mentovich, E., Belgorodsky, B., Gozin, M., Richter, S., & Cohen, H. (2012). Doped biomolecules in miniaturized electric junctions. J. Am. Chem. Soc., 134, 8468e8473. Misra, N., Martinez, J. A., Huang, S.-C. J., Wang, Y., Stroeve, P., Grigoropoulos, C. P., & Noy, A. (2009). Bioelectronic silicon nanowire devices using functional membrane proteins. Proc. Natl. Acad. Sci. USA, 106, 13780e13784. Park, H., Afzali, A., Han, S.-J., Tulevski, G. S., Franklin, A. D., Tersoff, J., Hannon, J. B., & Haensch, W. (2012). High-density integration of carbon nanotubes via chemical self-assembly. Nat. Nanotechnol., 7, 787e791.
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Rinaldi, R., Biasco, A., Maruccio, G., Cingolani, R., Alliata, D., Andolfi, L., Facci, P., De Rienzo, F., Di Felice, R., & Molinari, E. (2002). Solid-state molecular rectifier based on self-organized metalloproteins. Adv. Mater., 14, 1449e1453. Rinaldi, R., Biasco, A., Maruccio, G., Cingolani, R., Alliata, D., Andolfi, L., Facci, P., De Rienzo, F., Di Felice, R., Molinari, E., Verbeet, M., & Canters, G. (2003). Electronic rectification in protein devices. Appl. Phys. Lett., 82, 472e474. Ron, I., Sepunaru, L., Itzhakov, S., Belenkova, T., Friedman, N., Pecht, I., Sheves, M., & Cahen, D. (2010). Proteins as electronic materials: electron transport through solid-state protein monolayer junctions. J. Am. Chem. Soc., 132, 4131e4140. Tanford, C. (2004). Ben Franklin stilled the waves. Oxford: Oxford University Press. Ulman, A. (2001). Formation and structure of self-assembled monolayers. Chem. Rev., 96, 1533e1554. Willner, I., & Katz, E. (2000). Integration of layered redox proteins and conductive supports for bioelectronic applications. Angew. Chem. Int. Ed., 39, 1180e1218. Zhao, J. W., Davis, J. J., Sansom, M. S. P., & Hung, A. (2004). Exploring the electronic and mechanical properties of protein using conducting atomic force microscopy. J. Am. Chem. Soc., 126, 5601e5069.
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